Transcription is a pivotal point when cells control gene expression. Until recently, it was thought that the control of transcription in animal cells occurred primarily during formation of a preinitiation complex. The hsp70 gene of Drosophila was an apparent outlier and served as a paradigm for a small number of genes that regulated transcription after initiation by controlling the duration of a pause, which occurs in the promoter proximal region. However, through the use of new genomic techniques, genome-wide mapping of Pol II has revealed that thousands of genes in metazoans have Pol II paused in a region 20 to 50 nucleotides downstream from the transcription start. Numerous studies have documented the presence of Pol II at promoters in cells, but few have endeavored to investigate the mechanisms that regulate the pause. The overall goal of this A1 renewal application is to elucidate mechanisms that regulate promoter proximal pausing using Drosophila as a model system. We have reconstituted promoter proximal pausing in Drosophila nuclear extracts and this will provide a biochemical basis for investigating pausing. Also, we have developed a new technique called permanganate- ChIP-seq that will allow us to monitor the behavior of paused Pol II genome wide in response to various experimental conditions designed to probe mechanisms of pausing in vivo. The proteins, DSIF and NELF, have central roles in pausing and somehow cause Pol II to pause upon associating with the elongation complex. An enigmatic component of Pol II constituting the carboxyl terminal domain (CTD) of the largest subunit also somehow regulates pausing. Finally, our genomic analyses of paused Pol II has provided strong evidence for at least two distinct mechanisms of pausing. One mechanism is orchestrated by a DNA binding protein called GAGA factor. GAGA factor recruits NELF to promoters to facilitate binding of NELF to the elongation complex. Another mechanism is orchestrated by a DNA binding protein we call M1BP. M1BP- associated genes have strikingly ordered arrays of nucleosomes in their gene bodies, and our analyses indicate that pausing results from a collision between Pol II and the first nucleosome. To obtain insight into mechanisms of promoter proximal pausing, we aim to do the following: 1) Structure/function analyses of DSIF and NELF; 2) Mutational analysis of the Pol II CTD; 3) Functional analysis of M1BP-mediated transcription. The analyses of DSIF and NELF focuses on the roles of RNA binding domains in DSIF and NELF and a small region of DSIF that we found contributes to pausing. The analyses of the Pol II CTD explores the possibility that distinct regions of the CTD have specific functions in pausing. The functional analysis of M1BP focuses on our new discovery that M1BP binds a glutathione S-transferase and this glutathione S-transferase can act at a gene's promoter. The combination of biochemical and in vivo approaches that can be applied to Drosophila uniquely qualifies this project for obtaining significant insight into the mechanism of promoter proximal pausing, thereby having significant impact our fundamental understanding of gene regulation.